DESCRIPTION: (from abstract). Bacterial keratitis can result in blindness or a loss of visual acuity through a pathologic process that involves the interaction of bacteria, their products, and the host reaction to bacterial proteins. The infections are caused with almost equal frequency by Gram-positive (e.g., Staphylococcus or pneumococcus) or Grain- negative (e.g., Pseudomonas or Serratia) bacteria. While the outcomes of these bacterial infections are similar, research conducted in this laboratory has shown that the fundamental mechanisms differ dramatically. For Gram-positive bacteria, a hemolytic exotoxin is the prime mediator of tissue damage. In Pseudomonas or Serratia keratitis, bacterial proteases are essential for the intra-corneal stage of infection. The long-term goal of this research is to determine the molecular events mediating keratitis and to devise means to interrupt those reactions that result in irreversible tissue damage. The objectives of the present proposal are to determine the molecular properties of Pseudomonas protease IV, an extracellular enzyme associated with corneal virulence. This protease is produced by nearly all Pseudomonas ocular isolates and a mutant deficient in this enzyme is attenuated in animal models of keratitis. The PI's laboratory has very recently determined the DNA sequence of the gene for protease IV making possible for the first time detailed molecular biology studies. The specific aims are: 1) construct a plasmid that codes for functional protease IV and determine if the plasmid can augment the virulence of its host bacterium 2) identify the amino acids that comprise the active site of this enzyme providing the data will guide the development of a protease inhibitor, 3) prepare allele replacement mutants deficient in the protease IV gene and perform genetic rescue experiments to prove the relationship between protease IV and corneal damage, and 4) test synthetic peptides for use an immunogens in developing an immune state protective against the tissue damage associated with Pseudomonas keratitis. The results of these studies are anticipated to provide new knowledge upon which can be based chemotherapeutic or immunologic means to limit the severity of Pseudomonas corneal infections.